The candidate for this Clinical Investigator Award, Dr. Satish Singh, has completed his clinical training in gastroenterology and will have spent three years studying the physiology of intestinal transport with Dr. Henry Binder and the regulation of intracellular pH (pHi) with Dr. Walter Boron. During his research years, the candidate has mastered fluorescence imaging techniques for monitoring pHi. Adapting techniques for perfusing renal tubules, he also has succeeded in perfusing single colonic crypts. Dr. Singh is fully committed to a career in academic medicine. His long-term goal is to become an independent clinician/scientist, performing basic research in areas relevant to gastrointestinal disease. The research proposed focuses on the normal pHi physiology of colonic crypt cells, and how they have adapted to the unique acid-base environment created by products of bacterial fermentation. The colonic lumen contains high levels of ammonia, short-chain fatty acids, and CO2. These neutral buffers would be expected to readily permeate most cell membranes, and greatly affect pHi. Because maintaining a stable pHi is critical to normal cell function and proliferation, it is hypothesized that colonic crypt cells possess unique defenses for maintaining pHi in the unique colonic environment. This hypothesis will be tested by microperfusing the lumen of single crypts isolated from rabbit proximal colon. Preliminary pHi data on rabbit crypts suggest that the apical barrier is impervious to NH3, whereas the basolateral membrane has normal NH3 permeability. Preliminary pHi data on perfused crypts from human proximal colon indicate the presence of a Na-H exchanger. The first aim is to (i) determine if crypt cells possess an apical barrier to lipophilic, neutral acid-base products of bacterial fermentation, and (ii) attempt to breach the barrier to provide fundamental insight into its nature. The second aim, is to (i) characterize the acid-base transporters that regulate crypt cell pH and (ii) explore the mechanism by which crypts secrete HCO-3 into the lumen. The third aim, is to extend the analysis to human crypts, eventually obtaining these from colonoscopic biopsies. The goal is to determine key kinetic properties of the transporters which, in the future, might be compared to those of crypts from patients with colonic pathology. Thus, the proposed work has aspects of fundamental importance to cell physiology, GI physiology, and medicine. The environment in which Dr. Singh will continue his career development has unique strengths in both cellular acid-base physiology (Dr. Boron) and colonic physiology (Dr. Binder). Both sponsors have trained successful fellows. Dr. Singh's experimental activities are based in Dr. Boron's laboratory in the Department of Cellular and Molecular Physiology, which has great depth in the areas of ion channels/transporters and epithelial physiology. Dr. Binder will provide overall guidance on the cell physiology and pathophysiology of the colon, on the clinical aspects of the human studies, and on how to best develop the career of a clinician/investigator.